PROJECT SUMMARY RNA nanotechnology has progressed rapidly during the past several years. This nanotechnology includes the integration of multiple functional modules into one nanoparticle, of which the scaffolds, ligands, therapeutics, and regulators can be composed mainly or exclusively of RNA. We have constructed RNA nanoparticles of diverse size, shape, and stoichiometry displaying high chemical and thermodynamic stability and demonstrated their ability to harbor different functional groups, such as siRNA, miRNA, ribozyme, drug, and cancer targeting RNA aptamer. All functional modules retain their authentic folding and independent functionalities for specific cell binding, gene silencing, and cancer targeting in vivo. Upon systemic injection in tumor bearing mice, RNA nanoparticles bind to xenograft and metastatic tumors specifically and strongly with little to no accumulation in healthy vital organs and tissues 3-4 hours post-administration. The RNA nanoparticles are non-toxic and display favorable biodistribution and pharmacokinetic profiles. Our long-term goal is to promote RNA nanoparticles as a new generation of drug for the treatment of cancers in the clinic. The short-term goal of this project is to characterize the behavior of RNA nanoparticles in vitro and in vivo, with an aim to improve the efficiency for specific cell targeting, internalization and intracellular trafficking, favorable biodistribution without entrapment in liver, endosome escape, and tumor regression. These studies are based on three central hypotheses: (1) intracellular trafficking pathways and endosome escape are critical for effective cancer therapy; (2) biodistribution and pharmacological profiles of RNA nanoparticles are shape and size dependent; and, (3) immune responses elicited by RNA nanoparticles are highly dependent on RNA sequence, chemical modifications, size, shape, and stoichiometry. To address our goals, we will (1) systemically dissect the intracellular pathways taken by RNA nanoparticles and enhance their endosome escape capabilities; (2) inspect the pharmacokinetics (PK); pharmacodynamics (PD); and biodistribution of RNA nanoparticles with the goal of enhancing cancer targeting with minimal accumulation in healthy organs; and, (3) evaluate the immune responses of RNA nanoparticles to minimize non-specific side effects, as well as develop methods to stimulate the immune system by incorporating immuno-stimulatory modules to RNA nanoparticles for cancer immunotherapy. Upon completion of these pre-clinical studies, we will have identified several RNA nanoparticles with optimized shape, size, and stoichiometry displaying favorable safety profiles and high therapeutic efficacy to comply with FDA Investigational New Drug guidelines for initiating clinical trials.